A. Field of the Invention
This invention relates to a method for assaying aliphatic alcohols, aliphatic aldehydes or .omega.-carboxylic acid derivatives thereof using a novel .omega.-carboxyalcohol oxidase. More particularly the present invention relates to a novel .omega.-carboxyalcohol oxidase having the following biochemical properties.
1. Enzymatic action: catalyzing at least one of the following reactions a) and b) EQU a) R--CH.sub.2 OH+O.sub.2 .fwdarw.R--CHO+H.sub.2 O.sub.2 EQU b) R--CHO+O.sub.2 +H.sub.2 O.fwdarw.R--COOH+H.sub.2 O.sub.2
wherein R--CH.sub.2 OH is an aliphatic alcohol or a corresponding .omega.-carboxylic acid derivative, except methanol and ethanol, R--CHO is the corresponding aliphatic aldehyde or its .omega.-carboxylic acid derivative, and R--COOH is the oxidized form of R--CHO.
2. Substrate specificity: having substrate specificity on at least HO.sub.2 C--(CH.sub.2).sub.11 --OH, H.sub.3 C--(CH.sub.2).sub.11 --OH, H.sub.3 C(CH.sub.2).sub.9 --OH, H.sub.3 C--(CH.sub.2).sub.7 --OH and H.sub.3 C--(CH.sub.2).sub.5 --OH, and having no substrate specificity on methanol, ethanol or glycerol.
3. Utilization of coenzyme: no utilization of NAD and NADP.
B. Description of the Prior Art
The hitherto-known oxidase having substrate specificity for aliphatic acids is alcohol oxidase (EC.1.1.3.13) which catalyzes the following reaction to form aldehyde. EQU R'''--CH.sub.2 OH+O.sub.2 .fwdarw.R'''--CHO+H.sub.2 O.sub.2
Alcohol oxidase from Basidiomycete (Biochim. Biophys. Acta, 151: 330-342 (1968), Biochem. Biophys. Res. Commun., 20: 630-634 (1965)), H. polymorpha, a yeast grown in methanol, and Kloeckera sp. (Agr. Biol. Chem., 36: 2297-2306 (1972), Eur. J. Biochem., 36: 250-256 (1973), ibid., 64: 341-350 (1976), Agr. Biol. Chem., 36: 68-75 (1972)) are known. Alcohol oxidase from Basidiomycete has substrate (R'''--CH.sub.2 OH) specificity for C.sub.2-4 straight chain primary alcohol, allylalcohol or 2-propione-1-ol. Alcohol oxidase from H. polymorpha has substrate specificity for C.sub.1-4 straight chain primary alcohol, 2-propene-1-ol or 2-butene-1-ol, and alcohol oxidase from Kloeckera sp. has substrate specificity for C.sub.1-4 straight chain primary alcohol, 2-propene-1-ol, 2-butene-1-ol or allylalcohol.
Hence, alcohol oxidase from these origins generates aldehyde and has substrate specificity, at least as regards straight chain primary alcohol, for lower aliphatic alcohol or C.sub.4 and below.
Further, enzymes having substrate specificity for glycerol, e.g. glycerol dehydrogenase or glycerol-2-dehydrogenase (EC.1.1.1.6, EC.1.1.1.72, EC.1.1.1.156) and glycerol oxidase are known. These enzymes oxidize glycerol and generate dihydroacetone or glyceroaldehyde, and have no activity for catalyzing a reaction which generates a carboxylic acid derivative.
There is also known an oxidase which has substrate specificity for fatty acids having an .omega.-hydroxy group (such compounds may also be referred to as aliphatic alcohols having an .omega.-carboxy group, hereinafter designated .omega.-carboxyalcohols), and which oxidizes the said substrate to generate dibasic acid. The enzyme is known from animal tissue (Biochim. Biophts. Acta., 46: 45-50 (1961)) as well as from plant tissue (Methods in Enzymology, Vol. 71: pp. 411-420, 1981). These enzymes require the presence of coenzyme NAD or NADP to be active.
As hereinabove reviewed, prior known alcohol oxidases have substrate specificities for lower alcohols of C.sub.4 and below, at least as regards straight chain aliphatic primary or secondary alcohol, for glycerol, or for .omega.-hydroxy carboxylic acid in the presence of coenzyme NAD or NADP. An enzyme which does not require coenzyme, has no substrate specificity for straight chain primary alcohol of C.sub.2 and below, and requires oxygen to generate hydrogen peroxide, has never been known.